1. Field of the Invention
The invention herein relates to a novel human monoclonal antibody fragment (Fab), cloned by phage display that binds specifically to oxidized forms of low density lipoproteins (OxLDL) and not native LDL. More particularly, it relates to the use of the antibody for improved methods of diagnosis and treatment of atherosclerosis.
2. Description of Prior Art
Atherosclerosis is a chronic inflammatory disease that results from hyperlipidemia and a complex interplay of a variety of environmental, metabolic and genetic risk factors. The oxidation of low density lipoprotein (LDL) plays a central, if not obligatory role, in the atherogenic process. Early studies showed that acetylation of LDL greatly enhanced its uptake by macrophges and that the uptake occurred via xe2x80x9cscavenger receptorsxe2x80x9d which were distinct from the classical LDL receptor. Unlike most receptors, these scavenger receptors were not downregualted following uptake of OxLDL. Due to the excessive uptake of OxLDL and its associated lipid by the macrophages, the cells obtained a characteristic foam-like appearance. The appearance of such cells is one of the first hallmarks of atherosclerotic disease. Foam cells accumulate within the intima (under the endothelial lining) of the vessel walls where they become unstable and plaques, the hallmarks of more advanced disease. Inflammatory conditions develop leading to the development of complicated lesions.
There is much evidence that OxLDL contributes to atherogenesis by a number of mechanisms. The oxidation of polyunsaturated fatty acids in phospholipids of lipoproteins generates many breakdown products such as malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and other reactive moieties attached to oxidized phospholipids. Many of these intermediate products are highly reactive and can interact with lysine residues of associated proteins and phospholipids to generate various adducts. These adducts are known to occur in vivo and are immunogenic. In murine models of atherosclerosis, such as apo-E deficient mice (ApoExe2x88x92/xe2x88x92) mice, atherosclerosis is correlated with the development of high titers of autoanitbodies to varous oxidation specfic epitopes of OxLDL. The consequences of such cellular and humoral responses are still poorly understood, but under certain conditions they can clearly modify the natural history of the disease.
It is generally accepted that it is the composition of atherosclerotic lesions, in particular the content of lipids, OxLDL, foam cells, and smooth muscle cells, that determines their properties. Foam cells are often found in the sites of lesion that are susceptible to rupture. Activated macrophages recruited to clear the apoptotic and necrotic foam cells, as well as OxLDL, secrete factors that weaken the plaque. Human pathology studies have shown that atheromas containing a large necrotic core, thin fibrous cap and large numbers of macrophage/foam cells in the shoulder are more predisposed to plaque rupture and thrombosis. These lesions, which frequently appear as mild or moderate coronary stenoses in angiographic studies, are characterized pathologically as large atheroma with extensive lipid pools exceeding 40% of plaque areas. Angiography only provides a measure of arteial lumen, but fails to detect vessel wall pathology. Diagnostic methods that provide a measure of the overall extent of the atherosclerotic lesion, with an emphasis on OxLDL and lipid content, would therefore be desirable. Moreover, the lipid core of atheromas can be assumed to contain extensive oxidized lipids that accumulated within foam cells and set free when cells undergo necrosis and apoptosis.
Non-invasive detection of atherosclerotic lesions is currently not clinically feasable. The gold standard for diagnosing atherosclerosis is angiography which detects abnormal vessel lumen contours caused by encroaching atherosclerosis but does not directly identify abnormalities of the vessel wall. The widely recognized limitations of angiography include poor correlation with functional stenosis, interobserver and intraobserver variability, underestimation of the extent of disease because of diffusely atherosclerotic vessels, and arterial remodeling. B mode and ultravascular ultrasonography can detect intima/media thickening and calcification of vascular walls, but cannot clearly assess specific tissue characteristics. Electron beam computed tomography detects only calcium in vessel walls. Magnetic resonance imaging is still an investigational tool for the detection of plaque components.
Human studies have suggested that plaque rupture frequently occurs in nonangiographically significant lesions that contain abundant lipid-laden macrophages and large lipid pools within atheromas. Therefore imaging of atherosclerosis directed at lipid rich areas would be of value, not only in detecting the extent of lesion burden, but also in the detecting clinically silent but xe2x80x9cactivexe2x80x9d lesions. Previous radioscintographic imaging agents have been limited by poor specificity, low in vivo uptake in atherosclerotic plaque, and slow elimination from the circulation, resulting in poor lesion/background ratios. Various imaging agents have been used including radiolabeled LDL, fragments of apolipoprotein B, autologous platelet and antiplatelet antibodies, non-specific antibodies and Fc fragments, hematoporphyrin derivatives, and anti-malonic acid monoclonal antibodies (Mabs).
OxLDL specific antibodies have been isolated from human and rabbit atherosclerotic lesions which contain tightly bound IgGs that recognize epitopes of OxLDL in vitro and stains atherosclerotic lesions in vitro. Mouse hybridoma cell lines have been generated for the production of Mabs against OxLDL and the antibodies were found to bind specifically to oxidized, rather than native phospholipids. However all of the antibodies previously described were monospecific, binding to only one form of OxLDL. The EO series of mouse Mabs described by Palinski et al. (1996), were able to bind either OxLDL or MDA-LDL, not both. Similarly, MDA2 and NA59, mouse Mabs described in other studies, bind MDA-LDL and HNE-LDL respectively. Most importantly, these mouse antibodies are limited in their usefulness for human applications in vivo as they illicit an immune response that prohibits their repeated administration.
Hybridoma technology, which is widely used in generating murine Mabs, is less successful in producing human hybridomas. Epstein Barr Virus (EBV) may be used to immortalize human lymphocytes, however due to the wide variety of neoepitopes in OxLDL, acquisition of human Mabs to many different epitopes would be arduous. Furthermore, clones derived by this technique are frequently unstable and low secretors. Additionally, the EBV-transformants produce IgM antibodies, while anti-OxLDL antibodies can be both IgG and IgM isotypes.
Phage display combinatorial library technology provides a useful method to generate human Mabs (Barbas and Lerner, 1991; Huse, et al., 1989). The libraries made from lymphocyte mRNA may consist of up to 108 recombinants of monoclonal Fab repertoires. By displaying the library on a filamentous phage surface and panning against a model epitope, monoclonal Fab antibodies can be selected and analyzed for their immunological properties and biological activities. Fabs are ideal for use in both therapeutic and diagnostic methods as they can be produced in large quantities inexpensively and they are innately non-immunogenic. Additionally, they are not whole antibody molecules which can initiate a cascade of immune responses upon binding to their antigen.
The invention herein is the discovery of an antibody that binds to a novel epitope of OxLDL and MDA-LDL, but not native LDL, and its uses in imaging of atherosclerotic plaques, as a means for targeting therapeutics, and as a therapeutic itself or model structure for the development of novel therapeutics for the treatment of atherosclerosis.
The invention is the discovery of a cloned human monoclonal Fab isolated from a phage display library generated from mRNA from peripheral blood mononuclear cells (PBMC) from a patient who was found to have high antibody titers to MDA-LDL. After serial rounds of panning, a monoclonal IgG Fab antibody was isolated which bound specifically to both MDA-LDL and copper-induced OxLDL, but did not bind native LDL, as determined by both direct and competition binding assays. The Fab was found to bind specifically to atherosclerotic plaques, both in vivo and in vitro in human, mouse, and rabbit tissue. Furthermore, it was found to inhibit the uptake of OxLDL by macrophages, suggesting that the epitope on OxLDL defined by the Fab may be an important ligand for the macrophage scavenger receptors in normal clearance or atherogenesis. We have named the Fab IK17.
Additionally, the invention overcomes the deficiencies of prior art detection methods for atherosclerotic lesions by the use of IK17. The invention describes a new method to non-invasively image the atherosclerotic lesions themselves by the use of the use of a Fab conjugated to an appropriate molecule for detection. This further provides a means for particular discrimination of lipid rich components and oxidation rich components in vivo. The non-invasive nature of the imaging method using the invention reduces cost and risks to the patient allowing the method to be used as a means to monitor the effects of a treatment regimen, as well as a primary detection method. The imaging method disclosed herein is more sensitive than previous methods allowing for the detection of atherosclerosis, both coronary and non-coronary, before the occurrence of significant stenosis, allowing for earlier intervention. It also provides a means for observing the vessel itself and assaying the amount of lipid present in the lesion, providing a prognostic indicator and a method to grade the pathology of the lesion. It is a method to quantitatively monitor the effects of a treatment regimen as human antibodies will not induce an immune response. This type of surveillance cannot be performed with murine antibodies due to the potentially life threatening immune response to repeated administration of non-human antibodies.
The invention allows for the improvement of current therapeutics and the development of novel ones for the treatment of atherosclerosis. The Fab provides a means for targeting therapeutic agents to the site of the plaques by covalently linking a thrombolytic agent, antioxidant, antimetalloproteinase or other therapeutic agent to the antibody. Alternatively, IK17 itself, or small molecule analogs of IK17, could be used as drugs. IK17 is known to inhibit the uptake of OxLDL by macrophages, thus inhibiting the formation of foam cells. Inhibition of foam cell formation could decrease the deposition of lipids on the vessel wall and slow the progression of the disease.
The invention herein is the discovery of a human monoclonal Fab that we have named IK17 that binds specifically to both OxLDL and MDA-LDL, but not native LDL, and uses of the Fab in the improved detection and treatment of atherosclerosis. This is the first discovery of an antibody that recognizes two forms of modified LDL. IK17 was isolated from a phage display library prepared from RNA from PMNCs from a donor with coronary heart disease. It was found to be specific to Cu-OxLDL and MDA-LDL by a number of direct and competition binding assays using purified LDL. It was also found to be highly effective in a macrophage uptake assay, inhibiting the phagocytosis of both OxLDL and apoptotic cells. Additionally, the Fab was found to be useful for labeling atherosclerotic plaques, both in vitro and in vivo. Radioactively labeled IK17 injected into mice was found to co-localize to atherosclerotic plaques as determined by Sudan(trademark) staining.
IK17 was cloned from a combinatorial Fab library by methods known to those skilled in the art. Briefly, human plasma samples were screened for the presence of antibodies to OxLDL using a chemiluminescence assay (Hxc3x6rkkxc3x6 et al., 1996). A patient was identified as having a high antibody titer to MDA-LDL. PBMC were isolated from the patient and total RNA was extracted and used as a template to synthesize cDNA. The cDNA was used as a template for PCR amplification of the light and heavy chains, as described previously (Barbas and Lerner, 1991). Subsequently, 3 pairs of extension primers were used for secondary amplification to add restriction sites to each of the three classes of fragments, V-kappa, V-lambda, and VH.
PCR products of the expected size were cloned into the phage display vector pComb3H. The resultant phagemid DNA was transformed into XL-1 blue E. coli cells by electroporation. Clones were panned against MDA-LDL coated onto an ELISA plates. A suspension containing approximately 109-1010 (100 xcexcl) of recombinant phage was applied to each coated well and incubated at 37xc2x0 C. for 1 hour. After incubation, the wells were washed, once after the first round of panning or 10 times after subsequent rounds to remove unbound phage. Bound phage were eluted and used to infect bacteria for amplification by methods well known to one skilled in the art. After the final round of panning, phagemid DNA was prepared to remove gene III which anchors Fab on the phage surface, by endonuclease digestion and religation. The resultant products were transformed into XL1-blue cells to express soluble Fab by induction with isopropyl beta-D-thiogalactopyranoside (IPTG). Cell lysates were prepared and ELISA assays were performed to analyze Fab production and MDA-LDL binding activity. For subsequent experiments, selected monoclonal Fabs were purified using an IgG(Fab) affinity column by methods known to one skilled in the art.
Plasmid DNA containing the VH and VL genes of the Fab was isolated from cells and sequenced using an automated sequencer. Nucleotide sequences were analyzed using the EMBL/GenBank database. Analysis revealed that the repertoire of Fab of the invention light chain uses a v-kappa 3 family gene (Vg/38xcexa/L6) with the rearrangement to Jxcexa2. The repertoire of heavy chain uses a VH3 family gene, 3-23VH26c/DP47, with the rearrangement to JH4b.
The binding specificity of affinity purified Fab was studied by analyzing binding of the purified protein to MDA-LDL, Cu-OxLDL, and native LDL as well as a panel of unrelated protein and nucleic acid antigens, using both direct binding and competition assays. The binding of the Fab was found to be specific to MDA-LDL and Cu-OxLDL, with a preference for MDA-LDL with an affinity of 37 nM. The Fab did not bind significantly to 4-HNE-LDL, nor did it bind to non-specific MDA modified proteins. The Fab was capable of binding both the lipid and protein fractions of the Cu-OxLDL, but did not bind the native LDL, either whole or fractionated
The ability of the Fab to localize to atherosclerotic plaques makes it ideal for use in a method for detection of atherosclerotic lesions. The Fab can be produced easily and inexpensively in large quantities, as opposed to antibodies produced from hybridoma cell lines. Additionally, hybridoma lines may be unstable and decrease antibody expression levels over time. As there are no IgG type molecules in the E. coli in which the Fab is produced, purification can be carried out in a single affinity purification step. The antibody can be linked to radioisotopes, paramagnetic labels, echogenic liposomes, or other appropriate agents that can be detected by imaging methods, and injected into the host intravenously. After an appropriate time, imaging can be performed, either whole body for diagnostic purposes or locally at specific sites, such as carotid artery, in a quantitative manner to assess the hosts response to a treatment regimen.
LDL and apoptotic cells accumulate at the site of atherosclerotic lesions and likely contribute to the pathology of the disease. However, they could be exploited as a means for targeting drugs to lesions. Drugs for the treatment of atherosclerosis could be targeted to the appropriate site by linking them to the IK17, which in turn binds to its unique, oxidation specific epitope in the lesion. Such a method could be used to reduce the effective dose of drugs currently being used for atherosclerosis by targeting them to and retaining them at the site of lesions. Additionally it could be used to target therapeutic agents with desired activities that were found to be cleared to rapidly to be effective.
As noted above, it was demonstrated that in animal models of atherosclerosis, immunization with MDA-LDL could ameliorate the progression of the disease. IK17 could be administered as a protein or it could be administered using a gene therapy vector, as a means to ameliorate the progression of atherosclerosis.
As the sequence of IK17(SEQ ID 1 and SEQ ID 2) is cloned it could be easily manipulated for a number of purposes. The coding sequence for linker amino acids, such as lysine or cystiene could be added for modification of IK17 with imaging or therapeutic agents. The pharmacodynamic properties of the antibody could be changes to increase stability, plasma clearance and tissue uptake. The sequences of the antigen recognition region could be mutagenized and subjected to additional rounds of screening with phage display against different model compounds to identify other OxLDL binding antibodies.